Green Fluorescent Protein Vectors

Green fluorescent proteins provide a valuable, noninvasive approach for investigating biological events in living cells and tissues. Our Living Colors green fluorescent proteins (GFPs) include proteins suitable for fusion, cell labeling, and reporter studies; in cell lines, animal models, bacteria, and yeast. Like all of Clontech's fluorescent proteins, AcGFP1 and ZsGreen1 can be detected in cells without adding cofactors or substrates, which makes them ideal for use in live cell assays (1). The genes for both proteins have been human codon-optimized to enhance their translation in mammalian cells (2), and the proteins have been optimized for bright emission and fast chromophore maturation.

ZsGreen1: An Extremely Bright Green Fluorescent Protein

ZsGreen1 is a bright green fluorescent protein that was derived from a reef coral belonging to class Anthozoa (1), and has been modified for higher solubility, brighter emission, and rapid chromophore maturation (8–12 hours) compared to the unmodified protein. ZsGreen1 can be easily detected in Western blots by our wide array of Living Colors Antibodies.

On-Demand (DD) ZsGreen1 Reporter System

No more tradeoffs—perform reporter studies with both a low background and a broad dynamic range. The DD-ZsGreen1 Reporter System combines a bright fluorescent protein reporter (ZsGreen1) for high signal intensity, coupled with ligand-dependent ProteoTuner protein stabilization/destabilization technology to eliminate background. ZsGreen1 is expressed as a fusion with a ligand-dependent destabilization domain (DD). The DD rapidly targets the reporter protein for proteasomal degradation, guaranteeing a low reporter background signal at the start of your experiment. However, when the small, membrane-permeant ligand Shield1 is added to the sample, it binds to the DD and protects the reporter from degradation, so that it can accumulate. The DD-ZsGreen1 Reporter System is available in plasmid and lentiviral formats.

AcGFP1: A True GFP Monomer

AcGFP1 is an engineered, fluorescent mutant of the wild-type protein derived from the jellyfish Aequorea coerulescens. It is a true monomer that does not form dimers as has been reported for AvGFP and EGFP (3–4). AcGFP1 is a superior alternative to Aequorea victoria GFPs (EGFP and AvGFP), especially if you are looking for a true monomer for fusion applications (5). Its open reading frame has been human codon optimized to increase the translational efficiency of the AcGFP1 mRNA, which results in higher mammalian cell expression levels. The AcGFP1 protein is stable, allowing you to monitor fluorescence over extended periods of time. The chromophore matures rapidly and is readily detected 8–12 hours after transfection. As with all of our fluorescent proteins, AcGFP1 is well tolerated by mammalian cells, and has been successfully used to establish stably transfected, clonal cell lines.

AcGFP1 has an excitation maximum of 475 nm and an emission maximum of 505 nm. Its brightness and spectral properties allow AcGFP1 to be detected using existing filter sets for FITC. This includes standard factory-installed microscope filters as well as custom-tailored, optimized sets such as those available from Chroma Technology Corporation (see www.chroma.com for details). AcGFP1 can be easily detected in both Western blot and immunoprecipitation applications by our wide array of Living Colors Antibodies.

AcGFP1 has been widely validated as a fusion tag, using a wide variety of proteins with diverse functions and subcellular locations (Figure 1, Panel C). AcGFP1 is particularly suited for use in multicolor applications, e.g., to simultaneously visualize the subcellular localization of two proteins of interest. It also performs well in cell-based assays that monitor protein subcellular trafficking (Figure 1, Panels A and B). Cells expressing AcGFP1 are easily detected and sorted by flow cytometry.

Figure 1. Use of AcGFP1 for fusions and fluorescence microscopy applications. Panels A and B. Activation of Protein Kinase C alpha was monitored with Living Colors AcGFP1. Panel A. HEK 293 cells were stably transfected with a plasmid encoding AcGFP1 fused to PKC alpha. Panel B. Cells were induced with 1.5 µg/ml PMA for 3 min. The PKC alpha-AcGFP1 fusion moves from the cytosol to the plasma membrane, a result consistent with the known mobilization pattern of PKC alpha. Panel C. HeLa cells were transiently transfected with pAcGFP1-Actin and visualized by fluorescence microscopy.

A True Monomer

The monomeric nature of the AcGFP1 protein (26.9 kDa, calculated molecular weight based on amino acid sequence) has been confirmed by three independent methods:

Figure 2. AcGFP1 is a monomeric protein. Panel A. Recombinant AcGFP1 protein was analyzed by FPLC gel filtration chromatography. Overall protein absorbance (A280) and chromophore excitation (A477) of the eluted material were monitored simultaneously. AcGFP1 elutes from the column at a retention time corresponding to a molecular weight of 24 kDa. The calculated molecular weight of AcGFP1 is 26.9 kDa. Panel B. Recombinant AcGFP1 protein was analyzed by sucrose density ultracentrifugation using a continuous gradient. Panel C. Pseudonative gel analysis of proteins. The oligomeric structure of proteins is preserved during SDS PAGE analysis if samples are kept at 4°C and not boiled prior to loading on a gel. Boiled and unboiled recombinant proteins (7.5 µg) were separated by SDS PAGE electrophoresis (12% acrylamide). In both the boiled (denatured) and unboiled (nondenatured) samples, AcGFP1 runs as a uniform band of ~30 kDa due to their monomeric structure. The unboiled (nondenatured) DsRed-Express runs at a much higher molecular weight than its denatured (boiled) counterpart due to its oligomeric structure.

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